BIOC 20010

Thomas Dodd. Practical session 1 Wednesday 28th September 2011.

28/09/11

Experiment 1: Calibration.
Introduction pH meters require calibration before they can be used to accurately detect the pH of sample solutions. In order for the pH reader to have a scale upon which to base its readings, it must be calibrated against two solutions of known pH concentration, the first to set a benchmark for the reader to base its readings on and the second as a comparison benchmark, as the pH meter does not have its own stored reference pHs. As temperature will also play a factor in pH, it must be constantly measured, using a temperature probe which is also incorporated into the pH probe. Using this technique, we can ensure reliable pH readings from the probe, provided it is handled with care and stored in a buffer solution of pH 7 when not in use. Aims The aim of this experiment is to successfully set calibration points for a pH probe, using solutions of known pH concentration. Materials and Methods This experiment incorporates the use of a pH probe, which is also fitted with a temperature probe, deionized water, a waste beaker, phosphate pH 6.86 and calibration buffer (pH 9.18) Procedure The pH meter was plugged in and switched on. The first calibration solution of phosphate pH 6.86 was opened and the pH electrode and temperature probe were lowered into the container and stirred gently. The CAL button on the pH meter console face was pushed and the detection setting was set to 6.86. The screen flashed the message NOT READY for several seconds. It then read READY and the CFM (confirm) button was pushed allowing this calibration number of pH 6.86 to be stored. The electrode was removed from the solution and washed down with deionized water which was rinsed into the waste beaker to avoid cross contamination. The probe was then lowered into the second solution containing calibration buffer pH 9.18. The screen flashed the message NOT READY for several seconds before switching to ready. The CFM (confirm) button was pushed allowing the instrument to store the second calibration point. The pH electrode was washed down with deionized water and the rinsing added to the waste beaker. The pH electrode was then placed back into its storage solution. Results The pH probe was successfully calibrated and could now be used to measure the pH of solutions in the other experiments.

Discussion The use of a pH meter is obviously invaluable in practical laboratory conditions, as it removes the need to constantly take samples of a solution for the total solutions pH to be checked. The simplicity of a pH meter is also quite considerable, as it is a simple circuit that measures the voltage of the probe, which will vary depending on the pH it is placed in and then converts the voltage to pH using the Nernst equation

Experiment 2: The effect of buffer strength.

Introduction This experiment will demonstrate the ability of a buffer to drastically alter the [A-] of a solution at a steep point even through the volume that brought about this shift was quite minute and the pH does not vary greatly. At the point around the pKa of the solution, the addition of a small volume of buffer will result in a sharp shift in [A-] This experiment will be repeated twice, with a smaller concentration of buffer the second time, using a proportionally smaller volume of acid. In this manner, we can demonstrate that it is not the volume of the solution that plays a role in the [A-] shift; rather it is the addition of a buffer to the solution at around its pKa that will result in the shift of [A-]. Aims The aim of this experiment is to successfully measure the pH shift of a solution of known concentration and pH with the addition of an acid of known volume and pH and obtain a table of results which can be graphed in order to indicate the sharp change in the curve where the pH shift took place. Materials and Methods This experiment incorporates the use of a pH probe, deionized water, a waste beaker, a 50mL conical flask, a stock of 1M HCl, 100mM Tris buffer, 10mM Tris buffer, a waste beaker, a P 1,000 pipette and a P 200 pipette. Procedure 50 mL of 100 mM Tris Buffer solution was placed into a clean 50 mL conical flask. The pH probe was removed from its storage solution, washed with deionized water which was added to the waste beaker. The probe was then placed into the solution. The pH reading was allowed to settle and the screen read an initial reading of pH 9.1 0.5 mL of 1M HCl was then added to the conical flask using the P 1,000 pipette and the solution was gently swirled. The probe was placed back into the solution and the second reading was taken. This process was repeated a total of 10 times, by the end of which the pH of the solution had changed to a pH of 1.24. The conical flask was rinsed with deionized water and the rinsing were placed in the waste beaker. The pH probe was also rinsed with deionized water and the rinsing were added to the waste beaker. 50 mL of 10 mM Tris buffer was placed in the conical flask and the pH probe was placed in the beaker. The screen showed an initial reading of pH 9.09 0.05 mL of 1M HCl was then added to the conical flask using the P 200 pipette and the solution was gently swirled. The probe was placed back into the solution and and the second reading was taken. This process was repeated a total of 10 times, by the end of which the pH of the solution had changed to a pH of 2.29.

Results The following tables of results and accompanying graphs were taken from this experiment:

1M HCl = 1 mol/Litre Additions made at 0.05 ml = 0.0005 Litres Molecular weight of HCl= 36.5 36.5 X 0.0005 = 0.01825 g Addition of 0.0005 moles HCl.

100 mM Tris
Reading Initial 1st addition 2nd addition 3rd addition 4th addition 5th addition 6th addition 7th addition 8th addition 9th addition 10th addition pH mL HCl moles HCl 9.10 0.00 0 8.83 0.50 0.0005 8.58 1.00 0.0010 8.39 1.50 0.0015 8.17 2.00 0.0020 7.95 2.50 0.0025 7.67 3.00 0.0030 7.28 3.50 0.0035 5.30 4.00 0.0040 1.57 4.50 0.0045 1.24 5.00 0.0050

10 9 8 7 6 pH 5 4 3 2 1 0 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045 0.0050 Moles HCl pH

1M HCl = 1 mol/Litre Additions made at 0.05 ml = 0.00005 Litres Molecular weight of HCl= 36.5 36.5 X 0.00005 = 0.001825 g Addition of 0.00005 moles HCl.

10 mM Tris
Reading Initial 1st addition 2nd addition 3rd addition 4th addition 5th addition 6th addition 7th addition 8th addition 9th addition 10th addition pH mL HCl moles HCl 9.09 0.00 0.00000 8.64 0.05 0.00005 8.43 0.10 0.00010 8.22 0.15 0.00015 8.06 0.20 0.00020 7.81 0.25 0.00025 7.55 0.30 0.00030 7.14 0.35 0.00035 5.33 0.40 0.00040 2.65 0.45 0.00045 2.28 0.50 0.00050

10 9 8 7 6

pH

5 4 3 2 1 0 pH

Moles HCl

Discussion As discussed in the aims section this experiment demonstrated the sharp shift of pH as the concentration approaches the pKa of Tris buffer (8.06) becomes extremely pronounced, compared to the addition of the same volume of acid at proportionally distant values from the pKa. The equivalence point of this solution is reached when the number of moles of HCl added = 0.005/2 = 0.0025 moles, as [HA] = [A-] at this point. At the equivalence point, pH = pKa + log([A-]/[HA]), but when [A-] = [HA], log([A-]/[HA]) will = 1. Therefore at the equivalence point, pH = pKa. Questions Q 3.1What does this curve tell you about the effect of the buffer concentration? A: This curve tells us that the pH of a solution can shift rapidly before settling to a more steady increase or decrease in pH when protons are added or taken away from the solution. For example, if there is acid being introduced into a solution, a buffer will protonate A- to produce HA in order to absorb the acid. If there is base being introduced into a solution, a buffer will de-protonate HA to produce A- in order to absorb the base. Q.32 Where does the pH change the least per mole of acid added? A: Based on the data gathered from the experiment, experiment, the pH changed least upon the addition of 0.001 moles and 0.0001 moles of HCl. Q3.3 Draw the structure of Tris at pH 10.0? at pH 2.0?
HO

HO

NH2

Tris buffer neither protonated nor deprotonated.

HO

Tris buffer at pH 10.0, having been deprotonated in order to neutralise the base in the solution.

Tris buffer at pH 2.00, having protonated in order to remove H+ ions from the solution

H H O
+

H H O
+

NH2

O H

Q 3.4 What is the pKa of Tris? In which pH range does it work effectively as a buffer and why? A According to Wikipedia, Tris has a pKa of 8.06. Tris would work best as a buffer in more basic environments, as it would easily de-protonate in order to release free H+ ions which would be used to neutralize basic compounds being introduced into the system. It would not work as proportionally well in an acid solution, as it will not willingly accept H+ ions, compared to loosing them.

Experiment 3: Determination of pKa values.

Introduction Different compounds will have the ability to cope with pH changes to different extents. In this experiment, the compounds : Glycine, Cysteine and Imidazole will be used to demonstrate this fact. Additions of Sodium Hydroxide (NaOH) will be made at intervals of o.5 mL and the pH of each solution will be measured after each addition. By taking these readings, we will be able to determine the pKa values of each solution. As Glycine exists in a zwitterionic form as both a cationic carboxylic acid and an anionic amine when in solution, it will require two titrations in order to determine it two pKa values. Both forms are indicated below.

Cationic carboxylic acid H3N+CH2CO2H

Anionic amine H2NCH2CO2-

Aims The aim of this experiment is to successfully measure the pH shift of solutions of known concentration and pH with the addition of a base of known volume and pH and obtain a table of results which can be graphed in order to extrapolate the pKa values of each solution. A second titration will be required for Glycine during which acid will be added, in order to obtain both zwitterionic pKa values. Materials and Methods This experiment incorporates the use of a pH probe, deionized water, a waste beaker, a 50mL conical flask, a stock of 1M NaOH, 50 mL 0.01M Glycine, 50 mL 0.01M Cysteine, 50 mL 0.01M Imidazole , a waste beaker, a P 1,000 pipette and a stock of 1M HCl. Procedure Glycine 50 mL of 0.01 M Glycine was placed into a clean 50 mL conical flask. The pH probe was removed from its storage solution, washed with deionized water which was added to the waste beaker. The probe was then placed into the solution. The pH reading was allowed to settle and the screen read an initial reading of pH 1.8 0.5 mL of 1M NaOH was then added to the conical flask using the P 1,000 pipette and the solution was gently swirled. The probe was placed back into the solution and the second reading was taken. This process was repeated a total of 15 times, by the end of which the pH of the solution had changed to a pH of 11.21 Cysteine 50 mL of 0.01 M Cyseine was placed into a clean 50 mL conical flask. The pH probe was removed from its storage solution, washed with deionized water which was added to the waste beaker. The probe was then placed into the solution. The pH reading was allowed to settle and the screen read an initial reading of pH 5.0 0.5 mL of 1M NaOH was then added to the conical flask using the P 1,000 pipette and the solution was gently swirled. The probe was placed back into the solution and the second reading was taken. This process was repeated a total of 10 times, by the end of which the pH of the solution had changed to a pH of 11.50 Imidazole 50 mL of 0.01 M Imidazole was placed into a clean 50 mL conical flask. The pH probe was removed from its storage solution, washed with deionized water which was added to the waste beaker. The probe was then placed into the solution. The pH reading was allowed to settle and the screen read an initial reading of pH 5.0 0.5 mL of 1M NaOH was then added to the conical flask using the P 1,000 pipette and the solution was gently swirled. The probe was placed back into the solution and the second reading was taken. This process was repeated a total of 10 times, by the end of which the pH of the solution had changed to a pH of 11.01 Results The following tables of results and accompanying graphs were taken from these experiments:

In order to obtain [A-] values, the total concentration of NaOH added was divided by the total volume of the solution at this concentration. For example, at total volume = 50.5 ml, [NaOH] = 5x10-5 5x10-5/ 0.0505 L = 9.901 X 10-4 = [A-]. This calculation was repeated for all addition in order for find [A-] for all three titrations. The [HA] values were then obtained and the log of [A-]/[HA] was graphed agains the pH in order to find the intercept of the two axes. This would allow us to obtain the pKa values of the solutions. The following table was obtained in this manner in order to calculate the log [A-]/[HA] values. [HA] values and log values are not included where [HA] = 0. The following tables of information were obtained from measuring the pH values of each solution after each addition of NaOH.
Glycine Moles NaOH 0.00000 0.00005 0.00010 0.00015 0.00020 0.00025 0.00030 0.00035 0.00040 0.00045 0.00050 0.00055 0.00060 0.00065 0.00070 0.00075 Cystine pH 1.80 2.13 2.25 2.40 2.50 2.90 3.70 8.45 9.45 9.85 10.11 10.32 10.52 10.73 10.96 11.21 Reading Initial First Second Third Fourth Fifth Sixth Seventh Eighth Ninth tenth Moles NaOH 0 0.00005 0.0001 0.00015 0.0002 0.00025 0.0003 0.00035 0.0004 0.00045 0.0005 pH 5 7.93 8.4 8.74 9.07 9.44 9.55 10.52 10.95 11.24 11.5 Reading Initial First Second Third Fourth Fifth Sixth Seventh Eighth Ninth tenth eleventh twelfth Imidazole Moles NaOH 0 0.00005 0.0001 0.00015 0.0002 0.00025 0.0003 0.00035 0.0004 0.00045 0.0005 0.00055 0.0006

Reading Initial First Second Third Fourth Fifth Sixth Seventh Eighth Ninth tenth eleventh twelfth thirteenth Fourteenth Fifteenth

pH 5 5.1 6.02 6.45 6.6 6.83 7.14 7.28 7.49 7.73 8.07 8.41 11.01

Table of log[A-]/[HA]
Reading Initial First Second Third Fourth Fifth Sixth Seventh Eighth Moles NaOH 0.00000 0.00005 0.00010 0.00015 0.00020 0.00025 0.00030 0.00035 0.00040 Total volume 50 50.5 51 51.5 52 52.5 53 53.5 54 [A-] (mol/L) 0 9.901x10-4 1.961x10-3 2.913x10 3.846x10 4.762x10 5.66x10
-3 -3 -3

[HA] (mol L-1) 0.01 8.911x10-3 7.843x10-3 6.796x10 5.769x10 4.762x10 3.774x10 2.804x10 1.852x10
-3 -3 -3 -3 -3 -3

[A-]/[HA] 0 0.11111 0.25003 0.42864 0.66667 1 1.49974 2.3331 3.99946

log[A-]/[HA] N/A -0.95425 -0.60201 -0.36791 -0.17609 0 0.17602 0.36793 0.602

-3 -3 -3

6.542x10 7.407x10

Ninth tenth

0.00045 0.00050

54.5 55

8.257x10-3 9.091x10
-3

9.173x10-4 0

9.00142 N/A

0.95431 N/A

We can plot these values against the pH, and the point at which they cross the y axis will give us the pKa:

pKa values
12 10.52 10.93 11.24 9.44 8.45 7.14 7.28 7.49 7.73

10 8.4 8.74 9.07 8 6.45

9.44

9.95

pH

7.93

6.02 5.1

6.7 6.83 6

Glycine Cysteine

4 2.4 2.25 2 2.68 2.9

Imidazole 3.7

2.03

2.12

0 -1.5 -1 -0.5 0 0.5 1 1.5

log [A- ]/[HA]

Discussion By graphing the results we took from each titration, we can calculate approximate pKa values of each solution . As Glycine exists in a zwitterionic form, it would require two titrations, one with acid and one with base, in order to determine the pKa values of both forms. Due to time constraints, only the titration of an acidic solution of Glycine could be carried out. In actuality, the cationic and anionic pKa values are 2.34 and 9.6 respectively. As Imidazole and Cysteine exist in one form only in solution, they will return a single pKa values of 6.95 and 9.58 respectively. An interesting thing to note was that all of the solutions pKa values returned in this experiment were slightly higher than what they are in actuality. This suggests that perhaps there was some error in the pH calibration in, as the result was never lower than it should have been. The true pKa values for Glycine and cysteine were obtained from http://www.cem.msu.edu/~cem252/sp97/ch24/ch24aa.html Imidazoles pKa value was found from: http://en.wikipedia.org/wiki/Imidazole. Questions Q 4.1 Look up the pKa values for the compounds. How well do your pKa values agree with those found in literature? A The calculated values for each compounds pKa correlate quite well to the known values for each compound. A 2.4 pKa value was obtained for Glycine, when its actual pKa values is 2.35. 6.75 was obtained for Imidazole, whose actual pKa is 6.95. Cysteine returned a value of 9.58, with the true value being 10.25. While this is reasonably accurate, it is the least accurate of the three titrations. The result is probably down to human error, possibly impatience while waiting for the pH reading to settle, or incorrect cleaning of the pH probe between sample readings. Q4.2: Some of the compounds have multiple titrations. Which? A Glycine has multiple titrations due to being zwitterionic, as discussed before. Therefore it would require two titrations to detect both pKa values, as discussed before. Q4.3: For the compounds with multiple titrations: Are you able to observe the multiple titrations individually or are you getting an overall average? A When the full graph is plotted, an overall pKa value can be obtained, however when the graph is divided into its two components, the pKa for each functional group can be calculated.

Experiment 4: The analysis of Vinegar.

Introduction In this experiment, a sample of vinegar will be titrated against 1M of NaOH in order to determine he concentration of acetic acid in the sample. In order to detect the end point of the titration; the equivalence point, we will require the use of a colour indicator. Phenolphthalein will be used in this instance. Phenolphthalein is colourless in acidic solutions, but turns pink above a pH of 8.2. At, or very close to the equivalence point the indicator will turn the solution from colourless to pink. Aims

The aim of this experiment is to conduct two titrations on a sample of vinegar with additions of NaOH, the first to establish a general equivalence point and the second to establish a more accurate equivalence point. By observing the colour change that takes place at the equivalence point and noting the amount of NaOH that had been added to the vinegar sample, we can calculate the amount of acetic acid in the solution, since this reaction occurs in a 1:1 ratio. Methods and Materials 25 mL vinegar,a graduated cylinder,a conical flask, a sample of phenolphthalein, a waste beaker, a pipette, a stock of deionised water, a burette containing the 1 M NaOH and a white sheet of A4 paper. Procedure 25 ml of vinegar was placed in a clean conical flask and two drops of phenolphthalein were placed in the beaker using a pipette. The beaker was placed underneath the burette containing NaOH. It was ensured that the meniscus was at the graduation mark at eye level before proceeding. The tap of the burette was opened, allowing a steady stream of NaOH to enter the conical flask. When 10 ml of NaOH had been placed in the beaker, the tap was closed and the beaker was refilled back to its starting conditions. The tap was opened again, allowing the NaOH to flow through, although this time at a slower pace, so as to detect the end point. Traces of pink were detected in the conical flask, but the solution returned to colourless after a gentle swirl of the conical flask. A final end point of roughly 19.3 was detected as the point when the solution had reached its equivalence point. The contents of the conical flask were placed into the waste beaker and the burette was refilled to its starting point. As before, 25 ml of vinegar was placed into the conical flask and two drops of phenolphthalein were placed into the solution. This time, a more cautious addition of NaOH was practiced as the volume reached 19 ml. However the second reading gave a very sharp change of pink to colour less at almost exactly 21.2 ml NaOH. This was the figure chosen to calculate the amount of acetic acid present in the solution. Results As this reaction occurs in a 1:1 ratio, we can use the equation M1 X V1 = M2 X V2 to solve for the molarity of the acetic acid. The molar mass of acetic acid CH3CO2H is 60.05280 g/mol Titre 1: 19.3 ml M1 X V 1 = M 2 X V 2 1 X 19.3 = x x 0.25 19.3 = 0.25x X = 0.772 M 0.772 M X 60.0528 = 46.3608 g/L 46.3608 g/L = 4.63608 g/100ml present = 4.6% w/v

Titre 2: (accurate titration): 21.2 ml 1 X 21.1 = x x 0.25 21.1 = 0.25x

X=0.848 M.

60.0528 x 0.848 = 50.9247 g/L 50.92g/L = 5.092g/100ml present = 5.1% (w/v)

Discussion One possible reason for the second titre value being higher than the first in this experiment may have to do with the use of a graduated cylinder being used to measure the vinegar solution, which would have been somewhat inaccurate. Another factor may have been that myself and my lab partner took turns determining the end point each time. Both of these errors can be rectified by repeating the titration with a more cautios approach. Even though two different acetic acid concentrations were obtained, they both fall withinthe limits of regular diluted vinegar : (4% to 8% acetic acid). However, the second value is still to be taken as the true value of the sample tested, as a much more precise colour change was detected at this point.